Cancer is one of the most widespread diseases of mankind and a major cause of death worldwide. In an effort to find an effective treatment or a cure for one or more of the many different types of cancers and cancer disease, over the last couple of decades, numerous groups have invested a tremendous amount of time, effort and financial resources. However, to date, of the available cancer treatments and therapies, only a few offer any considerable degree of success.
Cancer is caused in many cases by the effects of oncoproteins. These are proteins having different structures as compared to their counterpart proteins in normal, healthy organisms. These oncoproteins are capable of transforming a normal cell into an uncontrollable, proliferating cell i.e., a cancerous cell, leading to the formation and growth of cancerous tumors. Oncoproteins are formed and expressed in an organism as a product of onco-genes, whose nucleotides sequence encodes the oncoprotein. Oncogenes occur as a result of a mutation of a “normal”, healthy gene, typically referred to as the “proto-oncogene.” Such mutations in proto-oncogenes produce protein products, which alter the normal programs of cell proliferation, differentiation and death. In a human cancer cell, one cell-signaling pathway in in which a proto-oncogene is mutated is the RAS-RAF-MEK-ER MAP kinase-signaling pathway. This pathway has been found to mediate cellular responses to growth signals. (Peyssonnaux et al., Biol. Cell, 93:53-62 (2001)).
The cell-signaling pathway involves the binding of a RAS substrate to activate a Raf kinase enzyme, which in turn activates the MEK kinase and so forth. There are three cytoplasmic serine/threonine RAF kinase proteins, which are polypeptides encoded by the nucleotide sequence of three Raf genes. The three Raf proteins found in mammals are A-Raf, B-Raf and C-Raf (C-raf is also known as Raf-1). (Biol. Cell, 93:53-62 (2001)). One feature in common among the three proteins is that they all share highly conserved regions, called CR1, CR2 and CR3. The CR1 domain is rich in cysteine residues, while the CR2 region contains many serines and threonine residues. The CR3 domain contains the kinase activity. The three naturally occurring Raf proteins also feature size differences. On average, B-raf proteins are larger than the other two, having a molecular weight of about 90 kDa, while the A-raf and C-raf have an average molecular weight of about 70 kDa. All three RAF proteins function by phosphorylating MEK-1/2, which in turn phosphorylates Erk-1/2, thereby activating the MEK-ERK MAP kinase portion of the signaling pathway described above. Structure, activity and function of the members of the Raf kinase family are further described in detail in Morrison and Cutler, Current Opinion in Cell Biology, 9:174-179 (1997) and U.S. Pat. Nos. 5,618,670, 5,156,841, and 6,566,581.
The B-raf protein has been found to be much more capable, than the A-raf and C-raf proteins, of phosphorylating the MEK-1 and MEK-2 proteins. More specifically, the B-raf phosphorylating activity is about 500× stronger than that of A-raf and about 10x stronger than that of C-raf. (Mol. Cell Biol.,15 (1997)). Accordingly, B-raf has become a potential target for regulating the RAS-RAF-MEK-ERK-MAP signaling pathway and, in turn, regulating programmed cell proliferation, cell differentiation and cell death.
B-raf kinase is commonly activated by somatic point mutations in cancerous cells. For example, B-raf somatic missense mutations occur in about 66% of malignant melanomas and at lower frequency in a wide range of human cancers. B-raf mutations have been found in 28 primary cancers/STC's, including 6 of 9 primary melanomas, 12 of 15 melanoma STC's, 4 of 33 colorectal carcinomas, 5 or 35 ovarian neoplasms, and 1 of 182 sarcomas. Although B-raf mutations occur in a wide range of cancers, there is a trend towards the occurance of mutations in cancer types in which a substantial portion of cases are known to harbor RAS mutations (for example, malignant melanomas, colorectal cancer, and borderline ovarian cancers). Mutated B-raf proteins have elevated kinase activity and are transforming in NIH3T3 cells. All mutations of B-raf have been found to be within the kinase domain, with a single substitution (V600E) accounting for about 80% of the mutated B-raf proteins discovered to date. It is worth noting that Ras function is not required for the growth of cancer cell lines with the V599E mutation. The high frequency of B-raf mutations in melanomas and the relative lack of effective therapies for advanced stages of this disease suggest that inhibition of wild-type B-raf and/or mutated B-raf activity may provide new therapeutic opportunities for metastatic and/or malignant melanomas.
Various groups have proposed different classes of compounds to generally modulate, or specifically inhibit, Raf kinase activity, for use to treat Raf-mediated disorders. For example, the PCT publication, WO 99/32106, describes substituted heterocyclic ureas for the inhibition of Raf kinase, WO 03/047523, describes methods for treating cancers resulting from the up-regulation of the RAF-MEK-ERK pathway using Gleevec® and “Gleevec®-like” compounds, WO 00/42012, describes delta-carboxyaryl substituted diphenyl ureas as Raf kinase inhibitors, WO 01/38324, describes substituted heteroaryl compounds for the inhibition of B-Raf kinase, U.S. Publication No. 2001/006975, describes methods of treating tumors mediated by raf kinase using substituted urea compounds, and U.S. Pat. No. 6,187,799, describes methods of treating tumors mediated by raf kinase using aryl urea compounds.